This invention was made with government support under NIMH grant MH-45614 awarded by the National Institutes of Health. The government has certain rights in the invention.
1. Field Of the Invention
The invention relates to dopamine receptors from mammalian species and the genes corresponding to such receptors. In particular, it relates to the human dopamine receptor D.sub.4. Specifically, the invention relates to the isolation, cloning and sequencing of the human D.sub.4 receptor gene. The invention also relates to the construction of eukaryotic expression vectors capable of expression of the human D.sub.4 dopamine receptor in cultures of transformed eukaryotic cells and the synthesis of the human D.sub.4 dopamine receptor in such cultures. The invention relates to the use of such cultures of transformed eukaryotic cells producing the human D.sub.4 dopamine receptor for the characterization of antipsychotic drugs.
2. Information Disclosure
Dopamine is a neurotransmitter that participates in a variety of different functions mediated by the nervous system, including vision, movement, and behavior. See generally Cooper, J. et al., "The Biochemical Basis of Neuropharmacology," 161-195 (Oxford University Press, NY 3d Ed. 1978). The diverse physiological actions of dopamine are in turn mediated by its interaction with two of the basic types of G protein-coupled receptors: D.sub.1 and D.sub.2, which respectively stimulate and inhibit the enzyme adenylyl cyclase. Kebabian, J. and Calne, D., Nature 277:93-96 (1979). Alterations in the number or activity of these receptors may be a contributory factor in disease states such as Parkinson's disease (a movement disorder) and schizophrenia (a behavioral disorder).
A great deal of information has accumulated on the biochemistry of the D.sub.1 and D.sub.2 dopamine receptors, and methods have been developed to solubilize and purify these receptor proteins. See Senogles, S. et al., Biochemistry 25: 749-753 (1986); Sengoles, S. et al., J. Biol. Chem. 263: 18996-19002 (1988); Gingrich, J. et al., Biochemistry 27:3907-3912 (1988); Gingrich, J. et al. (in press). The D.sub.1 dopamine receptor in several tissues appears to be a glycosylated membrane protein of about 72 kDa. Amlaiky, N. et al., Mol. Pharmacol. 31:129-134 (1987); Ninik, H. et al., Biochemistry 27:7594-7599 (1988). The D.sub.2 receptor has been suggested to have a higher molecular weight of about 90-150 kDa. Amlaiky, N. and Caron, M., J. Biol. Chem. 260:1983-1986 (1985); Amlaiky, N. and Caron, M., J. Neurochem. 47:196-204 (1986); Jarvie, J. et al., Mol. Pharmacol. 34: 91-97 (1988 ). Much less is known about a recently discovered additional dopamine receptor, termed D.sub.3. Sokoloff, P. et al., Nature 947:146-151 (1990). Dopamine receptors are primary targets in the clinical treatment of psychomotor disorders such as Parkinson's disease and affective disorders such as schizophrenia. Seeman, P. et al., Neuropsychopharm. 1:5-15 (1987); Seeman, P., Synapse 1:152-333 (1987). The three different dopamine receptors (D.sub.1, D.sub.2, D.sub.3) have been cloned as a result of nucleotide sequence homology which exists between these receptor genes. Bunzow, J. R. et al., Nature 336:783-787 (1988); Grandy, D. K. et al., Proc. Natl. Acad. Sci. U.S.A. 86:9762-9766 (1989); Dal Toso, R. et al., EMBO J. 8: 4025-4034 (1989); Zhou, Q-Y. et al., Nature 346:76-80 (1990); Sunahara, R. K. et al., Nature 34(5:80-83 (1990); Sokoloff, P. et al., Nature 347:146-151 (1990).
The antipsychotic clozapine is useful for socially withdrawn and treatment-resistant schizophrenics [Kane, J. et al., Nature 947:146-151 (1990)], but unlike other antipsychotic drugs, clozapine does not cause tardive dyskinesia [Casey, D. E., Psychopharmacology 99: 547-553 (1989)]. Clozapine, however, has dissociation constants at D.sub.2 and D.sub.3 which are 3 to 30-fold higher than the therapeutic free concentration of clozapine in plasma water [Ackenheil, M. et al., Arzneim-Forsch 2(5:1156-1158 (1976); Sandoz Canada, Inc., Clozaril: Summary of preclinical and clinical data (1990)]. This suggests the existence of dopamine receptors more sensitive to the antipsychotic clozapine.
We have cloned and sequenced such a human dopamine receptor which we term D.sub.4. The dopamine D.sub.4 receptor gene has high homology to the human dopamine D.sub.2 and D.sub.3 receptor genes. The pharmacological profile of this receptor resembles that of the D.sub.2 and D.sub.3 receptors but it has an affinity for clozapine which is tenfold higher. The present inventors envision that the D4 dopamine receptor disclosed as this invention may prove useful in discovering new types of drugs for schizophrenia that like clozapine do not induce tardive dyskinesia and other motor side effects.